Peptide fragments having cell death inhibitory activity

ABSTRACT

The present invention provides a peptide fragment or a series of peptide fragments having a cell death-inhibitory activity, having the amino acid sequence consisting of 103 amino acid residues at the C-terminal of selenoprotein P, or having said amino acid sequence with one or several amino acid residues therein being deleted, substituted or added, or having a partial sequence of either of the above amino acid sequences, a medicament for treatment comprising said peptide fragment or a series of peptide fragments, an antibody to said peptide fragment or a series of peptide fragments, and a method for screening a cell death-inhibitory activity using said peptide fragment or a series of peptide fragments. The preferable peptide fragment or a series of peptide fragments of the present invention has the amino acid sequences shown in SEQ ID NO: 1 and/or SEQ ID NO: 2 or has a partial sequence thereof.

This application is the national phase under 35 U.S.C. § 371 of PCTInternational Application No. PCT/JP99/06322 which has an Internationalfiling date of Nov. 12, 1999, which designated the United States ofAmerica and was not published in English.

The present invention relates to a protein having a novel function.Specifically, the present invention relates to a peptide fragment or aseries of peptide fragments having a cell death-inhibitory activity, aprocess for purifying said fragments, and an antibody to said peptidefragment or a series of peptide fragments. More specifically, thepresent invention relates to a peptide fragment or a series of peptidefragments that can be used as a medicament for protecting fromexacerbation of conditions of, preventing or treating various diseasessuch as diseases related to cell death, or as an additive allowing forproduction of useful material by inhibiting cell death in cell culture,and an antibody to said peptide fragments or a series of peptidefragments.

BACKGROUND ART

It has been suggested that cell death not only plays an important rolein basic control of the nervous system, the endocrine system and theimmune system in higher organisms but also is deeply involved in manydiseases (Thompson C. B., Science, vol. 267, p. 1456–1462 (1995). Somediseases including, for example, autoimmune diseases such as systemiclupus erythematosus, neurodegenerative diseases due to death of neurons,organ transplantation injuries associated with organ transplantation,etc. may be regarded as one due to influence of cell death whereapoptosis is involved.

Factors causing cell death includes both an extraneous factor and anintrinsic factor. For an extraneous factor, those of which substantialexistence as substance accelerating cell death have been establishedinclude TNF involved in the immune system (Zheng, L., et al., Nature,vol. 377, p. 348–351 (1995)), Fas ligand (Suda T., et al., Cell, vol.75, p. 1169–1178 (1993)), glucocorticoids (Wyllie A. H., Nature, vol.284, p. 555–556 (1980)), etc. An extraneous factor also includes lack ofa growth factor indispensable to cell growth, such as erythropoietin,interleukins, nerve growth factor, or lack of nutritional factors. Inthese cases, cell death is induced by apoptosis caused by change inphysiological conditions. Apoptosis may also be induced bynon-physiological stresses such as radiation, temperature, anticanceragents, calcium ionophore, active oxygen, etc. In addition, necrosis mayalso be induced by burn, toxic substance, ischemia, attack bycomplements, infection with virulent virus, administration of overdosemedicaments or overdose radiation.

For an intrinsic factor, there are changes in the metabolic system suchas intracellular concentration of Ca²⁺, metabolism of nucleic acids,metabolism of amino acids, metabolism of energy, etc., which lead tocell death. Control of these apoptotic signals could have lead toprotection from exacerbation of conditions of, prevention or treatmentof various diseases. However, at present, the mechanism is not so simplethat mere control of the causal substance and factors that have hithertobeen established cannot afford sufficient clinical application.

On the other hand, as substance that have hitherto been proved toinhibit cell death, intracellular factors such as bcl-2 and bcl-x areknown that are believed to inhibit most of apoptotic signals (Boise L.H., et al., Cell, vol. 74, p. 597–608 (1993)). However, theseagents—must intracellularly be expressed for causing inhibition of celldeath and effects can hardly be obtained by extracellular addition ofthese agents. Extracellular factors for inhibiting cell death have alsobeen reported that inhibit apoptosis by active oxygen, includingsuperoxide dismutase (hereinafter also referred to as “SOD”) (GreenlundL. J., et al., Neuron, vol. 14, p. 303–315 (1995)), catalase (SandstromP. A. and Buttke T. M., Proc. Natl. Acad. Sci. USA, vol. 90, p.4708–4712 (1993)), and glutathione peroxidase (Kayanoki Y., et al., J.Biochem., vol. 119, p. 817–822 (1996)). However, cell death cannoteffectively be inhibited by these extracellular factors alone.

While culturing cells, cell death is induced due to stress to cellsimposed by substances from the cultured cells per se or from extraneousadditives. However, it is not all the cells that are put to death undercertain conditions. For those cells that survived the circumstances,proteins necessary for suppressing the cell death-inducing signals dueto stress under their thresholds should have already been expressed, ornewly induced, either intracellularly or extracellularly. Such proteinsinclude, as envisaged, transcription factor, synthases, enzymes relatedto metabolism, oxidases, reductases, kinases, transferases,apoptosis-inhibiting proteins, etc. That is, sensitivity to stress ineach of respective cells may vary due to difference in their expressionlevel of these proteins. Thus, even if the mechanisms of cell death arenot always the same, if the cell death-inducing signals could besuppressed under their thresholds by extraneously adding an inhibitoryagent to cell death due to certain stress, then cell death couldpossibly be inhibited not only in cultured cells but also within theliving body where similar stress occurred.

Moreover, cell death is closely related to diseases. Thus,identification of a number of agents having a cell death-inhibitoryactivity within the living body to control a variety of cell deathswould not only allow for clinical application such as treatment ofdiseases but also for application to effective culture of culturedcells. Indeed, although some factors are known that inhibit cell death,e.g. bcl-2, bcl-x, etc. as intracellular cell death-inhibitory factors,or SOD, catalase, glutathione peroxidase, etc., as extracellularfactors, it is difficult to inhibit cell death in all types of cells byextracellular addition of these factors. This is due to difference inprocesses through which cell death is mediated based on difference intheir mechanisms. Taking this into consideration, there is a need toidentify activities that significantly, and more specifically, inhibit avariety of cell deaths. That is, for those cell deaths that are notsubject to inhibition by known materials, there is a need to searchingfor factors that can significantly inhibit said cell deaths. Inaddition, cell death-inhibitory factors are likely to be present formaintaining homeostasis within the living body and hence identificationof such factors is extremely significant.

While culturing cells under cell-free conditions or another specialconditions, apoptosis induced by stress is frequently observed. Cellculture is performed under these cell death-inducing conditions and withthe index of the cell death-inhibitory activities effective componentsin blood may be purified by using various chromatographies to therebyprepare proteinaceous components that inhibit cell death.

DISCLOSURE OF INVENTION

As a result of thorough investigation, it was found that a peptidefragment or a series of peptide fragments derived from said peptidefragment has an excellent cell death-inhibitory activity, said peptidefragment having the amino acid sequence consisting of 103 amino acidresidues at the C-terminal of selenoprotein P, or having said amino acidsequence with one or several amino acid residues therein being deleted,substituted or added, or having a partial sequence of either of theabove amino acid sequences. The term “a series of peptide fragments” asused herein refers to a group of peptide fragments with different minutestructures due to presence or absence of glycosylation, difference inelectric charge, diversity in fragmentation, etc.

Particularly preferable series of peptide fragments according to thepresent invention have the amino acid sequences of the formula (I):Lys Arg Cys Ile Asn Gln Leu Leu Cys Lys Leu Pro Thr Asp Ser Glu Leu AlaPro Arg Ser Xaa Cys Cys His Cys Arg His Leu (SEQ ID NO: 1) and/or theformula (II):Thr Gly Ser Ala Ile Thr Xaa Gln Cys Lys Glu Asn Leu Pro Ser Leu Cys SerXaa Gln Gly Leu Arg Ala Glu Glu Asn Ile (SEQ ID NO: 2)wherein Xaa represents selenocysteine, or a partial sequence of theseamino acid sequences.

Moreover, findings during their purification procedure revealed thatsaid peptide fragment or a series of peptide fragments (a) are recoveredin fractions of molecular weight 10 kDa to 30 kDa by molecular sizefractionation with membrane; (b) have structures showing isoelectricpoints at between pH 7 and pH 8 and at pH 8 or more in blood as a resultof testing of binding to an ion exchange resin; (c) show two bands atmolecular weight 13 to 14 kDa and two bands at 16 to 17 kDa as aglycosylated form of the former bands in non-reductive SDS-PAGE; and (d)in addition to the bands as described above, have a band pattern of 3 to4 kDa, 7 to 9 kDa and 10 to 12 kDa SDS-PAGE under reductive condition,and that said peptide fragment exhibits the activity even after furtherfragmentation.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows electrophoretograms either with silver staining or Westernblotting at various stages of purification of the peptide fragment or aseries of peptide fragments having a cell death-inhibitory activity ofthe present invention.

FIG. 2 shows electrophoretograms either with silver staining or Westernblotting denoting purity of the purified peptide fragment or a series ofpurified peptide fragments having a cell death-inhibitory activity ofthe present invention.

FIG. 3 shows electrophoretogram with silver staining denoting purity ofthe peptide fragment or a series of peptide fragments having a celldeath-inhibitory activity of the present invention, purified with acarrier column to which anti-selenoprotein P antibody is bound.

FIG. 4 shows electrophoretogram denoting behavior of the purifiedpeptide fragment or a series of purified peptide fragments having a celldeath-inhibitory activity of the present invention after treatment withN-glycosidase.

FIG. 5 shows electrophoretogram with silver staining denoting behaviorof the purified peptide fragment or a series of purified peptidefragments having a cell death-inhibitory activity of the presentinvention after reductive carboxymethylation.

FIG. 6 shows electrophoretogram with Western blotting denoting behaviorof the purified peptide fragment or a series of purified peptidefragments having a cell death-inhibitory activity of the presentinvention after reductive carboxymethylation.

FIG. 7 shows results of comparative experiment on the celldeath-inhibitory activity of the peptide fragment or a series of peptidefragments of the present invention with other proteins.

FIG. 8 shows results of comparative experiment on the celldeath-inhibitory activity of the peptide fragment or a series of peptidefragments of the present invention with other antioxidants.

FIG. 9 shows the cell death-inhibitory activity of the peptide fragmentor a series of peptide fragments of the present invention against celldeath induced by fatty acids.

FIG. 10 shows results of comparative experiment on the celldeath-inhibitory activity of the peptide fragment or a series of peptidefragments of the present invention with other antioxidants, typicallyvitamin E, against cell death induced by fatty acids.

BEST MODE FOR CARRYING OUT THE INVENTION

For screening factors having the cell death-inhibitory activity, aculture system needs to be established where cell death is induced. Asone of preferable embodiments in the present invention, a culture systemof Dami cells, human megakaryoblasts, with serum free culture mediumsupplemented with albumin was used for screening. Dami cells may besubcultured on a mixed culture medium of RPMI 1640, D-MEM and F-12(1:2:2) supplemented with 0.1% BSA and 0.05 μM 2-mercaptoethanol but canhardly grow on albumin-deprived medium. With culture medium containing0.01 to 0.5% human serum albumin, the cells grow normally but are put todeath on Day 4 abruptly not gradually. A diluted sample of activefractions may be added to this culture system to thereby estimate thecell death-inhibitory activity.

Although Dami cells might be most effectively used for assay, thepresent invention is not limited to Dami cells but any type of cells canbe utilized for screening the cell death-inhibitory activity insofar ascell death is induced under the similar conditions. Other applicablecell types include, for example, CEM, Molt4, etc. For albumin used inthis assay system, any albumin may be used insofar as cell death isobservable. By way of example, human serum albumin F-V (manufactured bySIGMA) may preferably be used.

Based on the assay system as described above, the present inventors haveaimed at components within the living body, especially those derivedfrom blood, and thoroughly investigated for searching the activity ofinterest. As a result, the present inventors have found a desiredactivity in plasma or serum from mammals, typically human beings.Fractions with detected cell death-inhibitory activity exhibited theactivity up to 1600-folds to 3200-folds dilution in case of plasma orserum from human source or the activity not more than 100-folds dilutionin case of fetal calf serum. For the purpose of quantification of thecell death-inhibitory activity as used herein, dilution of not more than100-folds is indicated as “0” while in case of dilution of more than100-folds, a figure of said dilution per se is used for indicating theactivity.

A series of peptide fragments, provided as active substance according tothe present invention, are rather stable to heat, a denaturing agent, abroad range of pH or protease in blood as compared to common enzymes andhence can be purified by using a wide variety of purificationprocedures. Thus, fractionations with applicable various carriers may beused such as various chromatographic procedures including heparinchromatography, cation exchange chromatography, anion exchangechromatography, hydrophobic chromatography, gel filtrationchromatography, reverse phase chromatography, hydroxyapatitechromatography, etc. In addition to these, other various fractionationsmay also be applicable such as ammonium sulfate precipitation, molecularsize fractionation with membrane, isoelectric focusing, electrophoreticfractionation, etc. These fractionations may suitably be used incombination to effectively fractionate the desired cell death-inhibitoryactivity. One of preferable combinations is shown in Example 2. Briefly,in the order of manipulation, it includes heparin chromatography,ammonium sulfate precipitation, anion exchange chromatography, cationexchange chromatography, hydrophobic chromatography, heparinchromatography, gel filtration chromatography, reverse phasechromatography and anion exchange chromatography.

This combination of purification procedures can afford active fractionof such purity as having not more than 5% estimated impurities, forexample, with the activity of 2×10⁵/1 mg protein/ml starting from humanplasma as a source. In view of the activity of starting plasma beingabout 20 to 40/1 mg protein/ml, it is estimated that specific activitywas increased by about 5,000 to 10,000 times.

The components having the cell death-inhibitory activity from plasmaaccording to the present invention purified and identified as describedabove are characterized by the following properties.

Heparin Binding

The components having the cell death-inhibitory activity are tested fortheir binding capability to heparin and it is revealed that thecomponents bind to heparin weakly. This finding suggests that thecomponents having the cell death-inhibitory activity according to thepresent invention are relatively charged positive. Thus, it is estimatedthat the components are possibly involved in protection of cellularsurface by binding with heparan sulfate on the surface of cells such aserythrocytes or the vascular endothelial cells easily subject to stressin blood.

Distribution of Activity by Molecular Size Fractionation with Membrane

For heparin-binding fractions from plasma, the cell death-inhibitoryactivity is concentrated with membranes of fractionating molecularweights 10 kDa, 30 kDa and 50 kDa to recover 90 to 95% activity for 10kDa, 10 to 20% for 30 kDa and 0 to 10% for 50 kDa. Thus, 80 to 90% ofthe components having the cell death-inhibitory activity according tothe present invention has a molecular weight of 10 kDa to 30 kDa in thepresence of other heparin-binding proteins. However, some of thecomponents having the activity have a molecular weight of more than thatrange, suggesting that there exist other active substances withdifferent molecular weights due to modification, polymerization ordifference in processing.

Fractionation with Ammonium Sulfate

For a sample of crude fractionation, all the active components areprecipitated with about 2 M ammonium sulfate. More strictly speaking,however, addition of about 3 M ammonium sulfate is necessary forprecipitating all the active components. The active components of thepresent invention are rather poorly salted-out although they mayoccasionally co-precipitate with some other proteins while salting out.

Binding to Ion Exchange Resin

With a suitable buffer of about 20 mM, the active components partiallybind to an anion exchanger at pH 8.0 or more but it is not all theactive components that bind. On the other hand, the active componentsalso bind to a cation exchanger at pH 7.0 or less. Thus, it is estimatedthat the active components of the present invention have both structureshaving isoelectric point at between pH 7 and 8 and having isoelectricpoint at pH 8 or more in blood.

Fractionation with Hydrophobic Chromatography

With Macro-Prep Methyl HIC or Macro-Prep t-butyl HIC carrier, adsorptionof the active fractions is hardly observed in the presence of 20 mMTris, pH 8.0, 200 mM NaCl and 1.2 M ammonium sulfate. When aconcentration of ammonium sulfate is increased to 1.5 M, however, 30 to50% of the active fractions are adsorbed. If a concentration of ammoniumsulfate is increased up to 2 to 2.4 M, almost all the active fractionsmay be adsorbed. With another carriers, it is possible to purifyefficiently the active components of the present invention under thesimilar conditions.

Fractionation with Gel Filtration

When the heparin-binding fractions are further fractionated by using gelfiltration chromatography, almost all the activity is recovered infractions of size 30 kDa to 40 kDa of molecular weight. On the contrary,the active components of the present invention, as actually obtained,have a molecular weight of 30 kDa or less in electrophoresis. Thus, itis estimated that the active components of the present invention arelikely to bind to other molecules.

Page (Polyacrylamide Gel Electrophoresis)

Fractionation of the active components of the present invention withPAGE under non-denaturing condition does not render the activity beingconverged to a single band. It is thus estimated that the activecomponents of the present invention are not represented by a singlestructure but exist in various forms with different molecular weightsdue to formation of dimer, difference in charge, glycosylation orvarious types of fragmentation of the peptide fragments consisting ofthe active components of the present invention. It is demonstrated inSDS-PAGE under non-reductive condition that the active components areconsisted of peptides showing two bands of molecular weight about 13 to14 kDa and two bands of about 16 to 17 kDa, the latter being aglycosylated form of the former. Under reductive condition, bands ofabout 3 to 4 kDa, about 7 to 9 kDa and about 10 to 12 kDa also occur inaddition to the above bands. This suggests that there are peptideshaving S—S bonds therein corresponding to the bands of about 13 to 14kDa and of about 16 to 17 kDa, wherein some of the peptides areinternally cleaved, and reduction cleaves the S—S bonds to producepeptides of the above additional sizes. This is supported by the factthat an antibody directed to the peptide fragment of about 3 to 4 kDa isreactive with all the peptide fragments other than those of about 7 to 9kDa and of about 10 to 12 kDa. Moreover, since the peptide fragment perse of about 3 to 4 kDa obtained under reductive condition still has thecell death-inhibitory activity, it is highly possible that this peptidefragment comprises a region deeply concerned with the activity.

Analysis of N-Terminal Amino Acid Sequence

It was found that the peptide fragments identified in the above PAGE hadhigh homology to the amino acid sequence consisting of 103 amino acidresidues at the C-terminal of human selenoprotein P as estimated fromcDNA of human selenoprotein P.

The N-terminal amino acid sequence was analyzed and, as a result, it wasfound that the peptide fragment having the cell death-inhibitoryactivity of the present invention or a series of peptide fragmentsderived from said peptide fragment had as a basic unit (1) a peptidehaving the amino acid sequence: Lys Arg Cys Ile Asn Gln Leu Leu Cys LysLeu Pro Thr Asp Ser Glu Leu Ala Pro Arg Ser Xaa Cys Cys His Cys Arg HisLeu (SEQ ID NO: 1), starting from the 260th Lys in human selenoproteinP, and (2) a peptide having the amino acid sequence: Thr Gly Ser Ala IleThr Xaa Gln Cys Lys Glu Asn Lys Pro Ser Leu Cys Ser Xaa Gln Gys Leu ArgAla Glu Glu Asn Ile (SEQ ID NO: 2) wherein Xaa is selenocysteine,starting from the 293rd Thr in human selenoprotein P.

The active components of the present invention are present as aconjugate or a complex of the above constituting units, i.e. the peptidefragments, and exert the cell death-inhibitory activity. Each of therespective constituting units has also the activity. There also existdiverse molecular species due to the presence or absence ofglycosylation, difference in charge, difference in fragmentation,especially diversity of the peptide fragments at the C-terminal end.Even in a mixture of such diverse molecular species, the celldeath-inhibitory activity of the present invention is exhibited.Therefore, the active components of the present invention encompass notonly each of the individual peptide fragments having the activity orpartial fragments thereof but also a group of the diverse peptidefragments, i.e. a series of peptide fragments, as a whole, insofar asthe cell death-inhibitory activity is exerted.

It has not been reported that there are processed forms of selenoproteinP with the sizes as in the present invention and much less that suggestssuch fragments alone have the activity.

Selenoprotein P was identified in 1977 as another selenium-containingprotein other than glutathione-peroxidase. In 1982, it was revealed thatselenium was incorporated into said protein in the form ofselenocysteine. In 1991, a full-length amino acid sequence ofselenoprotein P was determined by cloning selenoprotein P cDNA and, as aresult, possibility that said protein contains at most tenselenocysteine residues was demonstrated (Hill K. E. and Burk R. F.,Biomed. Environ. Sci., 10, p. 198–208 (1997)). However, there have beenno attempt to perform expression of a recombinant protein, or toidentify amino acid sequences corresponding to the active peptidefragment or a series of the peptide fragments of the present inventionin purified selenoprotein P, or to identify the active site. There is areport that human selenoprotein P was purified with anti-selenoprotein Pantibody. However, the antibody used therein recognized the amino acidsequence at the N-terminal of selenoprotein P and hence cannot be usedfor purifying the peptide fragment in accordance with the presentinvention. Therefore, the instant application is the first to estimatethe activity of the active peptide fragment or a series of the activepeptide fragments as characterized herein.

The activity of selenoprotein P has been reported including anantioxidant activity due to the presence of selenium and glutathioneperoxidase activity. Up till the present, however, there is no reportthat the peptide fragment or a series of peptide fragments obtained inaccordance with the present invention are indeed present within theliving body and exhibit the excellent activity. Of course, the presenceof the activity as characterized herein has not been reported.

Comparison of Activity with Other Proteins

Other than the peptide fragment or a series of peptide fragments havingthe cell death-inhibitory activity of the present invention,selenoproteins and related proteins with an antioxidant activity areexamined whether they exhibit the cell death-inhibitory activity in Damicells. As a result, the activity is somewhat observed only inglutathione peroxidase and SOD. However, in comparison with the peptidefragment or a series of peptide fragments having the celldeath-inhibitory activity of the present invention, the activity is aslow as 1/100 or less of that of the present invention, which may beregarded as substantially no activity. Then, compared with a full-lengthselenoprotein P, most relevant to the active components of the presentinvention, marked predominance of the active components of the presentinvention, fragmented product of selenoprotein P, is observed for thecell death-inhibitory activity and hence significance of “fragmentation”is demonstrated. That is, the active components of the present inventionas characterized herein are the only proteins having the celldeath-inhibitory C activity in blood that are not hitherto known.Therefore, identifying the presence of the active components has greatsignificance.

It is also possible to design chemically synthesized compounds, based onthe above finding, by utilizing the peptide fragment provided inaccordance with the present invention as a leading substance.

Using the peptide fragment having the cell death-inhibitory activity ofthe present invention as an immunogen, an antibody may be obtained thatrecognizes and binds to said novel peptide fragment. Although anymaterial containing the peptide fragment or a series of peptidefragments of the present invention may serve as an immunogen, thefraction prepared in Example 2 may preferably be used. The peptidefragment of the present invention or a portion thereof may also be usedas an immunogen that is prepared by using a peptide synthesizer orproduced from microorganisms such as E. coli or yeast with the geneticrecombination technique. Also, an expression plasmid for animal cell inwhich a gene encoding the peptide fragment of the present invention or aportion thereof is incorporated may be used as a DNA vaccine for animmunogen.

Such a peptide fragment for use as an immunogen preferably has, but isnot limited to, the amino acid sequence: Lys Arg Cys Ile Asn Gln Leu LeuCys Lys Leu Pro Thr Asp Ser Glu Leu Ala Pro Arg Ser Xaa Cys Cys His CysArg His Leu Ile Phe Glu Lys Thr Gly Ser Ala Ile Thr Xaa Gln Cys Lys GluAsn Leu Pro Ser Leu Cys Ser Xaa Gln Gly Leu Arg Ala Glu Glu Asn Ile ThrGlu Ser Cys Gln Xaa Arg Leu Pro Pro Ala Ala Xaa Gln Ile Ser Gln Gln LeuIle Pro Thr Glu Ala Ser Ala Ser Xaa Arg Xaa Lys Asn Gln Ala Lys Lys XaaGlu Xaa Pro Ser Asn, wherein Xaa is selenocysteine (SEQ ID NO: 3), orthe amino acid sequence: Lys Arg Cys Ile Asn Gln Leu Leu Cys Lys Leu ProThr Asp Ser Glu Leu Ala Pro Arg (SEQ ID NO: 4).

Although any mammals may be used for immunization, rabbit is preferablyused for obtaining antiserum and mouse is preferred if a monoclonalantibody is prepared by the cell fusion technique as described below.Age of animals may be, for example, 5 to 10 weeks in case of mouse. Bothmale and female animals may be used. An antigen for immunization may beused, for example, as a suspension in an appropriate adjuvant or as asolution in a physiological saline, which is then administered toanimals intraperitoneally, subcutaneously or intravenously. Thisimmunization is performed once to five times at the interval of 2 to 3weeks. The final immunization is made, for example, by suspending theantigen for immunization in physiological saline and intravenouslyadministering the suspension to animals. From the immunized animals,blood is drawn for preparing antiserum or spleen cells are prepared forobtaining hybridoma producing an antibody as described in Kohler G. andMilstein C., Nature, vol. 256, p. 495 (1975). In case of mouse, forexample, spleen cells from the immunized mice are fused with mousemyeloma cells to produce hybridomas.

Any culture medium may be used that is suitable for culturinghybridomas. Commonly, RPMI 1640 or Eagle MEM, supplemented with 5 to 10%fetal calf serum, 3.5 to 4.0 g/l L-glutamine and antibiotics such aspenicillin or streptomycin, is used. A serum free medium such as ASF 104(manufactured by Ajinomoto K. K.) or CM-B (manufactured by SankoJun-yaku K. K.) may also be used. Among the hybridomas obtained, thoseproducing a monoclonal antibody specific to the peptide fragment of thepresent invention are screened. Screening may be performed, for example,by sampling supernatants from the culture of hybridomas and examiningwhether they react with the peptide fragment of the present invention ora portion thereof, using the known techniques such as EIA, RIA orWestern blotting. This procedure may also be applied for investigatingwhether an antibody titer is increased in the immunized animals.

INDUSTRIAL APPLICABILITY

It is reported that in general a free fatty acid level is raised by asmuch as more than three times to bring about cellular toxicity at thetime when the living body is subject to a certain stress such ashemostasis, occurrence of inflammation, disorder in organs, damage incells, damage in blood vessels, bacterial infection, viral infection,etc. (“Chemistry of Lipids”, p. 170–179, ed. by Haruo Nakamura, AsakuraShoten (1990); “Handbook of Cerebral Apoplexy Experiment”, p. 437–471,supervised by Keiji Sano, IPC (1990)). From this teaching, it isanticipated that cells are adversely effected by a fatty acid undercircumstances where stressful state is maintained though sensitivity mayvary depending on the types of cells. That is, against stress that maybe encountered while operation, i.e. bleeding, hemostasis or ischemia,stress of reperfusion after ischemia associated with diseases or organtransplantation, or stress by continuous inflammation, the activepeptide fragment or a series of peptide fragments of the presentinvention, derived from selenoprotein P, can reduce adverse effects by afatty acid or prevent exacerbation of conditions by enhancingantioxidant activity of cells. The active peptide or a series of peptidefragments of the present invention would also serve as stabilizing cellsby enhancing antioxidant ability of cells against any phenomena whereoxidation stress of cells is raised through the similar mechanism.

It is anticipated that the active peptide fragment or a series ofpeptide fragments of the present invention function within the livingbody in the activated form after processing for protecting cells fromstress-derived death and for stabilizing cells. That is, when too muchstress is burdened to such a degree that the living body is no moredurable thereto, cell death could have occurred. Thus, in such a case,if the active peptide fragment or a series of peptide fragments of thepresent invention could be supplied extraneously, it would be possibleto prevent from progressing into severe diseases or to treat diseases.Diseases that can be prevented or treated by the active peptide fragmentor a series of peptide fragments of the present invention include onesinduced and effected by oxidation stress such as AIDS (acquiredimmunodeficiency syndrome), Parkinson's disease, Alzheimer's disease,etc. Since it is found that oxidized LDL is involved in onset ofarteriosclerosis as a causing factor, application for protection fromexacerbation of conditions, prevention or treatment of arteriosclerosisis also envisaged. It is also efficacious to diseases in whichreperfusion injury is observed such as myocardial infarction, cerebralinfarction or organ transplantation.

As for AIDS, close relationship between selenoproteins and AIDS or HIV(human immunodeficiency virus) has recently been reported. Briefly, itis reported that selenium level in serum is decreased with HIV infectionand that decrease in selenium level is correlated with mortality ordecrease in CD4 cells (Olmsted L. et al., Biol. Trace Elem. Res., vol.20, p. 59–65, 1989; Allard J. P. et al., Am. J. Clin. Nutr., vol. 67, p.143–147, 1998). It is also reported, that a nucleic acid sequence of HIVcomprises a sequence that encodes selenoproteins if frame-shifted andindeed, upon HIV infection, T lymphomas lose their ability to synthesizeselenoproteins, antioxidant enzymes such as glutathione peroxidase(Taylor E. W. et al., Biol. Trace Elem. Res., vol. 56, p. 63–91, 1997;Gladyshev V. N. et al., Proc. Natl. Acad. Sci. USA, vol. 96, p. 835–839,1999). It is also suggested that oxidation stress may be involved incell death of T lymphocytes in AIDS (Romero-Alvira D. et al., Med.Hypotheses, vol. 51, p. 169–173, 1998).

Actually, selenium level in blood was determined and, as a result, itwas found that the selenium level in blood in AIDS patients was as lowas about half of healthy adults. It was suspected that selenoprotein Plevel in blood in AIDS patients might possibly be different from that ofhealthy adults. Thus, selenoprotein P level in plasma was determined inAIDS patients with EIA. As a result, it was observed that aselenoprotein P level tends to be higher in AIDS patients with lessexacerbation of disease or with no onset of disease than in AIDSpatients with prompt exacerbation of disease. Moreover,immunoprecipitation was performed with a carrier to whichanti-selenoprotein P antibody is bound in order to compare the state ofselenoprotein P in plasma between AIDS patients and healthy adults. As aresult, it was demonstrated that AIDS patients with less exacerbateddisease or with no onset of disease had the same pattern as healthyadults whereas AIDS patients with promptly exacerbated disease showeddistinct pattern. From these results, possibility was suggested thatselenoprotein P might be useful for arresting and preventing onset ofAIDS.

Correlation between selenium level, especially selenoprotein P level, inblood and AIDS has been suggested as described above. However, thatpartial segments at the C-terminal of selenoprotein P having aparticular amino acid sequence have the cell death-inhibitory activitysignificantly higher than that of selenoprotein P per se and thus areuseful for prevention and treatment of AIDS has never known hitherto.

In addition, it is demonstrated that the active peptide fragment or aseries of peptide fragments efficiently work in culture of B cells and Tcells. Thus, they may also be used as an immunostimulatory orimmunoregulatory agent through stabilization or regulation of cells ofthe immune system. Moreover, they may also be used for enhancingefficiency of culture conditions, for example, in case of production ofuseful biological substance by protecting cells from death due toexcessive stress while cell culture.

The peptide fragment of the present invention or peptide fragmentshaving a partial sequence thereof and an antibody capable of binding tosaid peptide fragment may be used in an antigen detection system such asWestern blotting or ELISA and for preparing a diagnostic agent. Theantibody of the present invention may be bound to an appropriate carrierwhich is used for affinity chromatography for purifying the peptidefragment having the activity of the present invention. In addition, itis demonstrated that the active peptide fragment efficiently works inculture of B cells and T cells. A teaching that an antibody to theactive peptide fragment of the present invention as an immunogen doesaffect B cells was also obtained at immunization with said activepeptide fragment. Taken together these demonstration and teaching, theantibody of the present invention may also be used as animmunostimulatory or immunoregulatory agent through stabilization orregulation of cells of the immune system.

The present invention is explained in more detail by means of thefollowing Examples wherein reagents were purchased from Wako Jun-yaku K.K., Takara Shuzo K. K., Toyobo K. K. and New England BioLabs unlessotherwise instructed.

EXAMPLE 1

(Assay)

To 1 ml Dami cells (described in Greenberg S. M. et al., Blood, vol. 72,p. 1968–1977 (1988); 1×10⁶ cells/dish/3 ml), which can be subcultured inserum free medium SFO3 (manufactured by Sanko Jun-yaku K. K.) containing0.05 μM 2ME and 0.1% BSA, was added 2 ml 1:2:2 mixed medium (SA medium)of RPMI 1640/D-MEM/F-12. The cells were cultured for three days andrecovered for assay. The cells were washed twice with 50% PBS/SA/0.03%HSA (manufactured by SIGMA) and suspended in the same medium at 3×10⁴cells/ml. The cell suspension was added to a 96-well plate in each 200μl for wells for sample addition or in each 100 μl for wells for serialdilution. To the wells for sample addition was added 2 μl assay sampleand, after stirring, a serial dilution was made with the wellscontaining 100 μl cell suspension. The plate was incubated at 37° C. inCO₂ incubator for 4 to 5 days followed by estimation.

For estimation, it was examined to what folds of dilution of testedsamples the cells could survive in view of the fact that on Day 4 thecells in wells without the activity were put to death whereas the cellsin wells with the activity survived.

EXAMPLE 2

(Purification of Components Having Cell Death-Inhibitory Activity)

In the following purification procedure, the activity was estimated inaccordance with the assay procedure described in Example 1.

The cell death-inhibitory activity in plasma shows heparin-bindingactivity. Thus, fractionation with a heparin column was initiallyperformed for collecting heparin-binding fractions from plasma. Usinghuman plasma as starting material, heparin-binding proteins in plasmawere adsorbed to a heparin column (Heparin Sepharose: manufactured byPharmacia). After washing with 0.3 M sodium chloride, the adsorbedfractions were eluted with 2 M sodium chloride. Although most of thecell death-inhibitory activity of interest was recovered in thefractions after washing with 0.3 M sodium chloride, the fractions elutedwith 2 M sodium chloride were used for purification of active substance.

For crude fractionation of the heparin-bound cell death-inhibitoryactivity, fractionation with ammonium sulfate precipitation wasperformed. To the heparin-binding fractions eluted with 2 M sodiumchloride was added ammonium sulfate in an amount of 31.3% W/V (about 2M) based on a total amount of the fractions and precipitates wererecovered. The precipitates were dissolved in water and dialyzed againstwater with a dialysis membrane of M. W. 3,500 cut. After completion ofdialysis, the solution was recovered and 1 M Tris-HCl buffer, pH 8.0 wasadded thereto in an amount of 1/50 volume based on a total of thesolution. A concentration of the solution was adjusted with 20 mMTris-HCl buffer, pH 8.0 so that 20 to 30 of OD280 value was obtained.The solution was filtrated with 1.0 μm and 0.45 μm filters for removalof impurities.

An anion exchange chromatography was performed by passing theproteinaceous solution after filtration through anion exchangechromatographic carrier (Macro-prep High Q: manufactured by BioRad)equilibrated with 20 mM Tris-HCl buffer, pH 8.0. The activity wasdetected in non-adsorbed fractions and fractions eluted with 50 mMsodium chloride, which were collected. To the active fractions obtainedby anion exchange chromatography was added a 6:4 mixture of 1 M citratebuffer, pH 4.0 and 1 M citric acid in an amount of 1/50 volume based ona total of the fractions so that a proteinaceous solution was obtainedas 20 mM citrate buffer, pH about 4.0.

A cation exchange chromatography was performed by passing theproteinaceous solution through cation exchange chromatographic carrier(Macro-prep High S: manufactured by BioRad) equilibrated with 20 mMcitrate buffer, pH 4.0. The column was washed with 20 mM citrate buffer,pH 4.0 containing 220 mM sodium chloride. The activity was detected infractions eluted with 20 mM citrate buffer, pH 4.0 containing 550 mMsodium chloride, which were collected.

To the fractions eluted with 550 mM sodium chloride was added 1 MTris-Aminomethane solution in an amount of 1/30 volume based on a totalof the fractions and pH was adjusted to about 7.5. To this solution wasadded a 3.5 M ammonium sulfate solution (pH was adjusted to about 7.5 byadding 1 M Tris-HCl buffer, pH 8.5 in an amount of 1/50 volume) in anamount of ⅔ volume. Then, a salt concentration was adjusted so that 1.4M ammonium sulfate and 330 mM sodium chloride concentrations wereobtained. The solution was filtrated with 0.45 μm filter for removal ofimpurities.

A hydrophobic chromatography was performed by passing the proteinaceoussolution after filtration through hydrophobic chromatographic carrier(Macro-prep Methyl HIC: manufactured by BioRad) equilibrated with 20 mMTris-HCl buffer, pH 7.5 containing 1.4 M ammonium sulfate and 330 mMsodium chloride. The activity was detected in non-adsorbed fractions andfractions eluted with the buffer for equilibration, pH 7.5, which werecollected. The activity LF could hardly be detected in the adsorbedfractions. For the purpose of rendering the active fractions be adsorbedonto the hydrophobic chromatographic carrier, to the active fractionswas added the 3.5 M ammonium sulfate solution, pH about 7.5 so that 2.0M of an ammonium sulfate concentration was obtained. The sample waspassed through hydrophobic chromatographic carrier (Macro-prep MethylHIC: manufactured by BioRad) equilibrated with 20 mM Tris-HCl buffer, pH7.5 containing 2.0 M ammonium sulfate and 240 mM sodium chloride torender the active components be adsorbed. After washing with the bufferfor equilibration, the adsorbed active components were eluted with 20 mMTris-HCl buffer, pH 8.0. The recovered active fractions were dialyzedagainst water overnight. For ensuring adsorption of the active fractionsonto heparin column, 1 M citrate buffer, pH 4.5 was added to therecovered active fractions in an amount of 1/50 volume to adjust pHabout 5.0. Up to this procedure, see FIG. 1.

A 20 mM phosphate buffer, pH 6.5 (“Buffer A”) and a 20 mM phosphatebuffer, pH 6.2 containing 2 M sodium chloride (“Buffer B”) wereprepared. The pH adjusted, active fractions were passed through heparincolumn (Hi-Trap Heparin: manufactured by Pharmacia) equilibrated withBuffer A. The column was washed with a twice volume of a 5% mixture ofBuffer B in Buffer A (0.1 M NaCl). The active fraction was eluted with a20% mixture of Buffer B in Buffer A (0.4 M NaCl) and recovered. The thusobtained active fraction was concentrated to about 15 mg/ml with amembrane concentrator (Centriprep 3: manufactured by Amicon). To theconcentrated active fraction was added 2% acetic acid based on a totalof the fraction and then impurities were removed with 0.45 μm filter.

Gel filtration chromatography was performed by passing 1 ml of theactive fraction through gel filtration chromatographic carrier (Superdex200 pg: manufactured by Pharmacia) equilibrated with a solutioncontaining 2% acetic acid and 500 mM sodium chloride. Afterfractionation, the active fraction was recovered.

The above fraction was passed through C4 reverse phase HPLC (Wakosil5C4-200: 6 mm×150 mm: manufactured by Wako Jun-yaku K. K.) equilibratedwith 1% acetonitrile containing 0.1% trifluoroacetic acid and 1%isopropanol. The column was washed with the buffer used forequilibration. A linear gradient elution with 1% to 40% acetonitrilecontaining 0.1% trifluoroacetic acid and 1% isopropanol was thenperformed and the obtained active fractions were recovered. The progressof the activity and the specific activity obtained in each of the abovepurification procedures is summarized in Table 1 below.

TABLE 1 Purification Conc. of Protein Specific Step (mg/ml) ActivityActivity (1) 64 2400 38 (2) 22.6 12800 566 (3) 2.1 4800 2286 (4) 0.41600 4000 (5) 2.8 12800 4571 (6) 6.8 25600 3765 (7) 1.6 25600 16000 (8)0.9 204800 227556 (1): Starting plasma (2): Heparin elution/treatmentwith ammonium sulfate (3): Anion exchange chromatography; non-adsorbedfraction (4): Anion exchange chromatography; adsorption and elution (5):Cation exchange chromatography; adsorption and elution (6): Hydrophobicchromatography; adsorption and elution (7): HiTrap heparin, adsorptionand elution (8): C4 Reverse phase HPLC

For fractionating the obtained active fraction more fully, fractionationwas further performed using ion exchange chromatographic carrier Mini Q(manufactured by Pharmacia) A linear gradient elution with sodiumchloride was carried out under the condition of 20 mM ethanolamine, pH9.15. The activity was detected in all the fractions obtained, whichalso reacted with the antibody prepared in Example 4 as described below.This proved that the active substance was present in various differentstructures.

The active substance of interest at this stage, as a result ofelectrophoretic analysis, had under non-reductive condition severalbands at 10 kDa to 30 kDa and under reductive condition at least sixbands, i.e. each one band of smear at 3 to 4 kDa and at 7 to 9 kDa, twobands at 13 to 14 kDa, and two bands at 16 to 17 kDa. All these bandscould be detected in Western blotting analysis using the antibodydescribed in Example 4. A protein that reacted with the antibody wasalso detected at the vicinity of 28 to 29 kDa in electrophoresis undernon-reductive condition, suggesting that a dimer might possibly beformed. See FIG. 2.

EXAMPLE 3

(Analysis of N-Terminal Sequence of Active Components)

Amino acid sequence analysis with a gas phase sequencer revealed thatthe active components of the present invention consisted of a peptidecomprising the amino acid sequence: Lys Arg Cys Ile Asn Gln Leu Leu CysLys Leu Pro Thr Asp Ser Glu Leu Ala Pro Arg Ser Xaa Cys Cys His Cys ArgHis Leu (SEQ ID NO: 1) and a peptide comprising the amino acid sequence:Thr Gly Ser Ala Ile Thr Xaa Gln Cys Lys Glu Asn Leu Pro Ser Leu Cys SerXaa Gln Gly Leu Arg Ala Glu Glu Asn Ile, wherein Xaa is selenocysteine(SEQ ID NO: 2). A ratio of these peptides was in a range of from 1:1 to2:1 as estimated from an amount of amino acid residues recovered whilesequencing of this fraction. A recovery of amino acid residues fromother proteins than these two peptides was 5% or less. These twopeptides were separated by gel filtration chromatography and C4 reversephase HPLC under reduced condition to suggest the presence of molecularspecies formed by S—S bonding.

Among the peptides separated by C4 HPLC under reduced condition, apeptide having a molecular weight of 3 to 4 kDa, as a result ofsequencing analysis, had the amino acid sequence: Lys Arg Cys Ile AsnGln Leu Leu Cys Lys Leu Pro Thr Asp Ser Glu Leu Ala Pro Arg Ser (SEQ IDNO: 5) and the fraction consisted mainly of 7 to 9 kDa had the aminoacid sequence: Thr Gly Ser Ala Ile Thr Xaa Gln Cys Lys Glu Asn Leu ProSer Leu Cys Ser Xaa Gln Gly Leu Arg Ala Glu Glu Asn Ile (SEQ ID NO: 2).Both the fractions of 13 to 14 kDa and of 16 to 17 kDa had also theamino acid sequence: Lys Arg Cys Ile Asn Gln Leu Leu Cys Lys Leu Pro ThrAsp Ser Glu Leu Ala Pro Arg Ser (SEQ ID NO: 5). These amino acidsequences corresponded to the fragments starting from the 260th lysineand from the 293rd threonine in the amino acid sequence shown in thefollowing Table. 2, following the signal sequence, deduced from the cDNAsequence of human selenoprotein P previously published (Hill K. E. etal., Proc. Natl. Acad. Sci. USA, vol. 90, p. 537–541 (1993)).

TABLE 2 Met Trp Arg Ser Leu Gly Leu Ala Leu Ala Leu Cys Leu Leu Pro SerGly Gly Thr (signal sequence) Glu Ser Gln Asp Gln Ser Ser Leu Cys LysGln Pro Pro Ala Trp 15 Ser Ile Arg Asp Gln Asp Pro Met Leu Asn Ser AsnGly Ser Val 30 Thr Val Val Ala Leu Leu Gln Ala Ser Xaa Tyr Leu Cys IleIle 45 Glu Ala Ser Lys Leu Glu Asp Leu Arg Val Lys Leu Lys Lys Glu 60Gly Tyr Ser Asn Ile Ser Tyr Ile Val Val Asn His Gln Gly Ile 75 Ser SerArg Leu Lys Tyr Thr His Leu Lys Asn Lys Val Ser Glu 90 His Ile Pro ValTyr Gln Gln Glu Glu Asn Gln Thr Asp Val Trp 105 Thr Leu Leu Asn Gly SerLys Asp Asp Phe Leu Ile Tyr Asp Arg 120 Cys Gly Arg Leu Val Tyr His LeuGly Leu Pro Phe Ser Phe Leu 135 Thr Phe Pro Tyr Val Glu Glu Ala Ile LysIle Ala Tyr Cys Glu 150 Lys Lys Cys Gly Asn Cys Ser Leu Thr Thr Leu LysAsp Glu Asp 165 Phe Cys Lys Arg Val Ser Leu Ala Thr Val Asp Lys Thr ValGlu 180 Thr Pro Ser Pro His Tyr His His Glu His His His Asn His Gly 195His Gln His Leu Gly Ser Ser Glu Leu Ser Glu Asn Gln Gln Pro 210 Gly AlaPro Asn Ala Pro Thr His Pro Ala Pro Pro Gly Leu His 225 His His His LysHis Lys Gly Gln His Arg Gln Gly His Pro Glu 240 Asn Arg Asp Met Pro AlaSer Glu Asp Leu Gln Asp Leu Gln Lys 255 Lys Leu Cys Arg Lys Arg Cys IleAsn Gln Leu Leu Cys Lys Leu 270 Pro Thr Asp Ser Glu Leu Ala Pro Arg SerXaa Cys Cys His Cys 285 Arg His Leu Ile Phe Glu Lys Thr Gly Ser Ala IleThr Xaa Gln 300 Cys Lys Glu Asn Leu Pro Ser Leu Cys Ser Xaa Gln Gly LeuArg 315 Ala Glu Glu Asn Ile Thr Glu Ser Cys Gln Xaa Arg Leu Pro Pro 330Ala Ala Xaa Gln Ile Ser Gln Gln Leu Ile Pro Thr Glu Ala Ser 345 Ala SerXaa Arg Xaa Lys Asn Gln Ala Lys Lys Xaa Glu Xaa Pro 360 Ser Asnwherein Xaa is selenocysteine (SEQ ID NO:6)

EXAMPLE 4

(Preparation of Antibody to Active Components)

In order to prove that the bands obtained in Example 2 were derived fromone and same substance, a polyclonal anti-peptide antibody and amonoclonal antibody were prepared as described below. As a result ofWestern blotting using these antibodies, all the bands observed inelectrophoresis were recognized by the same monoclonal antibody and theanti-peptide antibody to prove that the peptide fragments had theidentical, though not uniform, structure.

{circumflex over (1)} Preparation of Anti-Peptide Antibody

For preparing an anti-peptide antibody, the active fraction wassubjected to gel filtration and C4 reverse phase HPLC under reducedcondition to prepare peptides of 3 to 4 kDa. Then, based on analysis ofthe amino acid sequence of said peptides, a peptide of 20 amino acidresidues was synthesized and used for immunization of rabbit.Specifically, a peptide having the sequence NH₂—Lys Arg Cys Ile Asn GlnLeu Leu Cys Lys Leu Pro Thr Asp Ser Glu Leu Ala Pro Arg-COOH (SEQ ID NO:4) was synthesized with a peptide synthesizer and purified by C18reverse phase HPLC. The purified peptide was bound to KLH at a ratio 1:1using glutaraldehyde and 200 μg of the conjugate was inoculated to twoNew Zealand white rabbits. Immunization as priming was made oncesubcutaneously at the back in the presence of Freund's completeadjuvant. Thereafter, three immunizations with subcutaneous inoculationat the back at each two weeks interval followed in the presence ofFreund's incomplete adjuvant and the animal were bled. Antiserum wasexamined for its reactivity with the immunogen by EIA and increase inantibody titer up to as high as 40,000-folds was demonstrated. Thisantiserum was affinity-purified with a carrier wherein the antigen wasbound to agarose to give an anti-peptide antibody that showed thesimilar reactivity to the antiserum.

{circumflex over (2)} Preparation of Monoclonal Antibody

For immunization as priming, 50 μg of the purified fraction of theactive components of the present invention as described in Example 2 wasonce inoculated intraperitoneally to Balb/c mice in the presence ofFreund's complete adjuvant. Thereafter, the mice were twice immunizedintraperitoneally in the presence of Freund's incomplete adjuvant at twoweeks interval. A week later, the mice were inoculated intravenouslywith 50 μg of the purified fraction of the active components. Three daysafter the final immunization, spleen cells were removed from the mice inthe conventional manner. Among five mice tested, the spleen cells fromtwo mice showed stronger reactivity with the immunogen in Westernblotting using the antiserum but were reduced in number to as low as1/10 than normally observed, suggesting that the antibody to immunogenmight have affected B cells.

The obtained spleen cells were mixed with myeloma cells P3X63Ag8.U1(P3U1) (ATCC deposit No. CRL-1597: Curr. Top. Microbiol. Immunol., vol.81, p. 1 (1978)) at a ratio of 1:1 to 1:2 and centrifuged (1,500 rpm, 5minutes). Supernatant was discarded and the precipitated cell pellet wassufficiently loosened and thereto was added 1 ml of a polyethyleneglycol solution (45% polyethylene glycol 4000, 55% RPMI medium),previously heated to 37° C., while stirring. After incubation at 37° C.for 5 minutes, RPMI medium was slowly added to make a total of 50 ml.After centrifugation (1,300 rpm, 7 minutes), supernatant was discardedand the cell pellet was moderately loosened. Thereto was added 50 ml ofEscron CM-B medium (manufactured by Sanko Jun-yaku K. K.) and the cellswere moderately suspended with a measuring pipette. Each 100 μl of thecell suspension was distributed to each well of four or five 96-wellcell culture plates and incubated in CO₂ incubator with 5% carbonic acidgas at 37° C. On the next day, each 100 μl HAT medium (Escron CM-Bmedium supplemented with 1×10⁴ M hypoxanthine, 1.5×10⁻³ M thymidine and4×10⁻⁷ M aminopterin) was distributed to each well and incubated in CO₂incubator with 5% carbonic acid gas at 37° C. In a descending order ofgrowth of hybridoma colonies, the culture medium was replaced with HTmedium (the HAT medium from which aminopterin was deprived). A portionof the culture supernatant was taken for screening hybridomas ofinterest by means of the following screening procedures, which consistedof a combination of EIA and Western blotting as described below.

(1) EIA

The synthetic peptidic antigen prepared as described above or purifiedantigen (2 μg/ml of protein) was added to a 96-well microtiter plate at50 μl/well and the plate was incubated at 4° C. overnight. The plate wasadded with 300 μl of 1% BSA (bovine serum albumin) solution andincubated similarly for masking. To the thus preparedantigen-immobilized plate was added culture supernatant of thehybridomas prepared by cell fusion and of the hybridomas after cloning.The plate was incubated at 4° C. for 1.5 hour, washed with PBS threetimes, and added with a solution of peroxidase-conjugated anti-mouseimmunoglobulin (manufactured by Kappel, diluted by 5,000-folds) at 100μl/well. After incubation at 4° C. for 1 hour, the plate was washed withPBS five times. To the plate was added a TMBZ substrate solution todevelop in the conventional manner and absorbance was measured at 450nm. As such, hybridoma clones that reacted with the purified antigenwere screened. Sixteen positive colonies were screened from about 500hybridomas.

(2) Western Blotting

Screening by Western blotting was performed for the positive colonies inEIA. The purified antigen was electrophoresed on 17.5%SDS-polyacrylamide gel and transferred to PVDF membrane. The membranewas excised into 0.4 to 0.5 cm width. Each strip was immersed into theculture supernatant solution of hybridomas and incubated at 37° C. for 1hour. Strip was then washed with TBST containing 0.05% Tween three timesand incubated in a 1:2000 dilution of alkaline phosphatase-conjugatedanti-mouse IgG (manufactured by TAGO) at 37° C. for 1 hour. Afterwashing with TBST three times, strip was developed with a color reagentusing BCIP/NBT (manufactured by Bio-Rad) and hybridomas showing coloredbands of the purified antigen were screened and cloned. The sameprocedures were employed for the hybridomas after cloning. The abovescreening provided two hybridoma clones that produced the desiredmonoclonal antibody.

EXAMPLE 5

(Purification of Selenoprotein P Fragment Using Anti-Selenoprotein PAntibody-Bound Carrier Column)

Heparin Sepharose-binding fraction from plasma was precipitated with 2 Mammonium sulfate. The precipitate was dissolved in more than 5 volumesof 20 mM Tris buffer, pH 8.0. Selenoprotein P present in this solutionwas adsorbed to an anti-selenoprotein P antibody-bound carrier column inwhich the anti-selenoprotein P antibody as described in Example 4 wasbound to a carrier. The carrier was washed with phosphate bufferedsaline (PBS) and selenoprotein P was eluted with 20 mM citrate buffer,pH 4.0 containing 4 M urea. The eluate was adsorbed to a cationexchanger (Macroprep High S, BioRad) equilibrated with 20 mM citratebuffer, pH 4.0. Then, gradient elution was performed with a saltconcentration of sodium chloride and a fraction of selenoprotein Pfragment having the cell death-inhibitory activity was recovered. Atthis stage, a full-length selenoprotein P could also be obtained butshowed the cell death-inhibitory activity per proteins that was muchlower than that of the fragment thereof. According to the procedures asdescribed herein, purification may be carried in a short time and henceselenoprotein P fragments could be obtained with higher celldeath-inhibitory activity per proteins. The fragments obtained at thisstage were also a fraction of a mixture containing various molecularspecies with varied sizes depending on the presence or absence ofglycosylation, intermolecular bonding, or inner cleavage, etc. They werea group of selenoprotein P fragments that showed a size ranging from 10to 30 kDa in electrophoresis under non-reductive condition. See FIG. 3.

EXAMPLE 6

(Treatment of Mini Q Active Fraction with N-Glycosidase)

In order to investigate the presence of glycosylation in the activefraction, the active fraction obtained by mini Q fractionation wastreated with N-glycosidase F to cleave N-type glycosylation, if any. Thetreatment was done in 150 mM Tris, pH 7.4. As a result, it was provedthat the two peptides at 16 to 17 kDa shifted to the size of 13 to 14kDa. No significant change was observed for the other peptides. See FIG.4.

EXAMPLE 7

(Reductive Carboxymethylation)

For obtaining more detailed information, the peptide fragment or aseries of peptide fragments of the present invention were subjected toreductive carboxymethylation followed by separation with reverse phaseC4 HPLC. The obtained peptide fragments were electrophoresed andanalyzed for their amino acid sequences. Electrophoresis revealed thattwo peptides, a size of which was expected to be 7 to 9 kDa prior totreatment, shifted to a distance corresponding to 10 to 12 kDa due toreductive carboxymethylation. It was proved by the reactivity with theanti-peptide antibody that only two peptide fragments, i.e. the peptidefragment of F3 and the fragment of F2 of 16 to 18 kDa, which wasexpected to show a molecular weight of 10 to 12 kDa prior to treatment,did not comprise the peptide fragment having the amino acid sequence:Lys Arg Cys Ile Asn Gln Leu Leu Cys Lys Leu Pro Thr Asp Ser Glu Leu AlaPro Arg (SEQ ID NO: 4).

As a result of amino acid sequence analysis, the peptide fragmentscontained in the above F2 and F3 fractions had the amino acid sequence:Thr Gly Ser Ala Ile Thr Xaa Gln Cys Lys Glu Asn Leu Pro Ser Leu Cys SerXaa Gln (SEQ ID NO: 7) wherein Xaa was selenocysteine. The F2 band at 16to 18 kDa shifted to the F3 band upon treatment with N-glycanase tothereby prove that the F2 band was a glycosylated form of the F3 band.All the fractions that were reacted with the anti-peptide antibody,including the fragments with peptides, a molecular weight of whichshifted upon N-glycanase treatment, had the amino acid sequence: Lys ArgCys Ile Asn Gln Leu Leu Cys Lys Leu Pro Thr Asp Ser Glu Leu Ala Pro ArgSer (SEQ ID NO: 5).

Taken the results obtained above and in Example 3 together, among thepeptide fragments corresponding to each of (1) 3 to 4 kDa, (2) 7 to 9kDa, (3) 10 to 12 kDa, (4) 13 to 14 kDa and (5) 16 to 17 kDa, of theactive substance, it is recognized that the bands (1), (4) and (5) arefragments starting from the 260th lysine whereas the bands (2) and (3)are fragments starting from the 293rd threonine. From the fact that thepeptide fragments (1), (2) and (3) were not obtained under non-reductivecondition, it was proved that these peptide fragments were formed afterinner cleavage of the peptide fragments (4) and (5) having unitstructures bound through S—S bonding. It was also proved that thepeptide fragment (5) was a glycosylated form of (4) viewing that theband (5) shifted to the band (4) upon N-glycanase treatment and that theband (5) was recognized by the antibody to the band (1). Moreover, bandsof different sizes, not derived from glycosylation, were detected at thevicinity of each band, and hence, it was estimated that several otherpeptide fragments with different size derived from the C-terminalexisted. See FIGS. 5 and 6.

EXAMPLE 8

(Comparison of Activity with Other Proteins)

Selenoproteins and related antioxidant proteins, not belonging to theactive components of the present invention, were examined for their celldeath-inhibitory activity. Glutathione peroxidase (manufactured bySIGMA) as antioxidant selenoprotein, glutathione reductase (manufacturedby Oriental Yeast K. K.), glutathione S transferase (manufactured bySIGMA), and superoxide dismutase (manufactured by Seikagaku Kogyo K. K.)as other related antioxidant proteins were used to test their celldeath-inhibitory activity in Dami cells for comparison. Each 70 μM ofthe samples was employed for measurement. Assay revealed some activityobserved for glutathione peroxidase and superoxide dismutase. However,in comparison with the peptide fragment and a series of peptidefragments having the cell death-inhibitory activity of the presentinvention as characterized herein, they showed the activity as low asabout 1/100 of the present invention. This obviously demonstrated thesuperiority of the peptide fragment and a series of peptide fragmentshaving the cell death-inhibitory activity of the present invention intheir activity. See FIG. 7.

In addition, the full-length selenoprotein P prepared with the antibodyaffinity column as described above was estimated for its celldeath-inhibitory activity in the same assay system for comparison. Itwas demonstrated that the peptide fragment and a series of peptidefragments of the present invention, a fragmented form of selenoproteinP, had the specific activity superior to that of the full-lengthselenoprotein P by more than 80-times, proving significance of“fragmentation”. See Table 3 below.

TABLE 3 Protein conc. Specific Sample (μg/ml) Activity activityFull-length 40 <100 <2500 selenoprotein P Peptide fragment 10 2000200000 of the invention

EXAMPLE 9

(Comparison of Activity with Other Antioxidants)

It was estimated to what extent vitamin E, known as being useful as anantioxidant to lipid oxidation, and catalase, acting for removal ofhydrogen peroxide, could inhibit cell death induced in the assay systemof the present invention while serum free culture in the presence ofHSA.

To 1 ml Dami cells (1×10⁶ cells/dish/3 ml), which can be subcultured inserum free medium SFO3 (manufactured by Sanko Jun-yaku K. K.) containing0.05 μM 2ME and 0.1% BSA, was added 0.2 ml 1:2:2 mixed medium (SAmedium) of RPMI 1640/D-MEM/F-12. The cells were cultured for three daysand recovered for assay. The cells were washed twice with 50%PBS/SA/0.03% HSA (manufactured by SIGMA) and suspended in the samemedium at 3×10⁴ cells/ml. The cell suspension was added to a 96-wellplate in each 190 μl for wells for sample addition or in each 100 μl forwells for serial dilution.

To the wells for sample addition was added each 10 μl of assay sample,i.e. 20 μM vitamin E, catalase or the selenoprotein P fragment and,after stirring, a serial dilution was made with the wells containing 100μl cell suspension. The plate was incubated at 37° C. in CO₂ incubatorfor 4 to 5 days followed by estimation. For estimation, a sampleconcentration necessary for cell death inhibition was compared to eachother on Day 4 and thereafter. It was demonstrated that catalase showedno cell death-inhibitory activity whereas vitamin E inhibited cell deathup to 125 nM but could not at 60 nM. The selenoprotein P fragment couldinhibit cell death up to 60 pM. From the fact that vitamin E did inhibitcell death in the assay system of the present invention, it wasestimated that peroxidization of fatty acids bound to HSA (SIGMA) mightbe responsible for cell death induction. Moreover, it was expected thatthe selenoprotein P fragment, which inhibited cell death moreeffectively than vitamin E, would also act much more efficiently toevents to which vitamin E was know to be effective. See FIG. 8.

EXAMPLE 10

(Inhibitory Activity to Cell Death Induced by Fatty Acid)

Any long-chain fatty acid with at least two double bonds including, forexample, eicosadienoic acid, dihomo-γ-linolenic acid, docosadienoicacid, docosatrienoic acid, adrenic acid, eicosapentaenoic acid,docosahexaenoic acid, linoleic acid, linolenic acid and arachidonic acidinduced cell death at 10 μM in serum free culture in the absence ofselenoprotein P. Among these, the most potent cell death inducer,arachidonic acid, linoleic acid and linolenic acid were thoroughlyinvestigated for their concentration that induced cell death as well asa concentration of selenoprotein P necessary for inhibiting the celldeath.

To 1 ml Dami cells (1×10⁶ cells/dish/3 ml), which can be subcultured inserum free medium SFO3 (manufactured by Sanko Jun-yaku K. K.) containing0.05 μM 2ME and 0.1% BSA, was added 2 ml 1:2:2 mixed medium (SA medium)of RPMI 1640/D-MEM/F-12. The cells were cultured for three days andrecovered for assay. The cells were washed twice with SA/0.05% fattyacid free BSA (manufactured by Wako Jun-yaku K. K.) and suspended at3×10⁴ cells/ml in the same medium containing 2 to 16 μM arachidonicacid, linoleic acid or linolenic acid. The cell suspension was added toa 96-well plate in each 198 μl for wells for sample addition or in each100 μl for wells for serial dilution.

To the wells for sample addition was added each 2 μl of 100 μM assaysample and, after stirring, a serial dilution was made with the wellscontaining 100 μl cell suspension. The plate was incubated at 37° C. inCO₂ incubator for 4 to 5 days. Cell death induction and inhibition ofcell death by selenoprotein P fragment were estimated with 1 μMselenoprotein P and effective concentration thereof by serial dilution.

It was demonstrated that cell death was induced in serum free culture ofthe cells in the presence of 4 μM or more multivalent unsaturated fattyacids such as arachidonic acid or linoleic acid and was completelyinhibited by 1 μM selenoprotein P fragment. See FIG. 9. As compared tovitamin E which inhibited cell death in the presence of 4 μM linoleicacid at an effective concentration of about 100 nM, a full-lengthselenoprotein P and selenoprotein P fragment inhibited at an effectiveconcentration of about 100 nM and 10 μM, respectively. Thus, theselenoprotein P fragment could inhibit cell death at much lowereffective concentration. See FIG. 10. From the fact that vitamin E, anantioxidant, did inhibit cell death, it was estimated that fatty acids,upon being peroxidized either intracellularly or extracellularly,damaged cells leading to cell death whereas the selenoprotein P fragmentefficiently prevented these events from occurring.

Then, various enzymes related to oxidation/reduction were investigatedfor their activity to inhibit cell death induced in Dami cells in thepresence of 4 μM linoleic acid or linolenic acid. The enzymes testedinclude glutathione peroxidase, superoxide dismutase, glutathionereductase, glutathione-S-transferase, and catalase. Only glutathioneperoxidase could inhibit cell death at 250 nM or more in the presence oflinoleic acid and at 500 nM or more in the presence of linolenic acid.The other enzymes, however, could not inhibit cell death even at 1 μM ormore. The fact that the selenoprotein P fragment could inhibit celldeath at as low as 10 pM in the same assay condition proved prominentefficacy of the selenoprotein P fragment. By varying a concentration offatty acids in induction of cell death, influence of fatty acids tovarious types of cells with different sensitivity or influence ofselenoprotein P thereto could be observed. Usually, cell death isinduced by the addition of 20 μM linoleic acid and, if cell death is notinduced by this condition, selenoprotein P is likely to be expressed.Using this system, the inhibitory activity to cell death induced byfatty acids can be estimated in various types of cells. This system wasconsidered to reflect the similar events to cell death induced by addingHSA (SIGMA).

Hitherto, the selenoprotein P fragment of the present invention wasproved to be effective in megakaryoblasts cell lines (Dami), Tcell-derived cell lines (Molt4, CEM, Jurkat), B cell-derived cell lines(P3X63AG8.653, P3X63AG8.U1), liver-derived cell lines (HepG2), nervoussystem-derived cell lines (IMR 32), kidney-derived cell lines (CRL1932), etc. Thus, it was highly expected that the selenoprotein Pfragment could also exert cell death-inhibitory activity to the cellsfrom the immune system, the nervous system or the hemopoietic system, orfrom the organs.

EXAMPLE 11

(Effect of Cell Death-Inhibitory Substance as Additive to Cell Culture).

Various cell lines including megakaryoblasts strains: Dami, hepatocytecell strains: HepG2, uterus-derived cell strains: Hela, kidney-derivedcell strains: CRL 1932, histic lymphocyte-derived cell lines: U937, Tcell-derived cell lines: Jurkat, Molt4 and CEM, fibroblast-derived celllines: L929, monocyte-derived cell lines: THP-1, B cell-derived celllines: P3X63AG8.653 and P3X63AG8.U1, and nervous system-derived celllines: IMR32 were cultured in RPMI 1640/D-MEM/F-12 (1:2:2) free fromtransferrin, insulin and sodium selenite in the presence or absence ofthe selenoprotein P fragment. It was demonstrated that exacerbation ofcellular conditions was not observed or at least inhibited in thepresence of the selenoprotein P fragment in all the types of cellstested. See Table 4. Moreover, in the presence of transferrin andinsulin, the cellular conditions could be maintained in all the types ofcells tested. Additional presence of 0.05% BSA was found to be moreefficacious.

When Jurkat cells were cultured in the presence of 5% human serumwherein selenoprotein P had perfectly been removed with a carrier towhich anti-selenoprotein P antibody was immobilized, growth of saidcells was exacerbated and decrease in intracellular glutathioneperoxidase activity, a kind of intracellular antioxidant enzyme, wasobserved. However, addition of the selenoprotein P fragment to thisculture system could restore the cellular growth and the glutathioneperoxidase activity to normal level. A similar effect could also beobserved for sodium selenite but the effect of the selenoprotein Pfragment was much excellent. Ebselen, a kind of selenium compounds, hadno equivalent effect. Thus, it was demonstrated that the selenoprotein Pfragment could replace sodium selenite and be used as additives to cellculture.

TABLE 4 Effect of selenoprotein P fragment in serum free culture P3X63AP3X63A Dami HepG2 HeLa CRL1932 U937 Jurkat Molt4 CEM L929 THP-1 G8.653G8.U1 IMR32 SeP (+) ⊚ ◯ ◯ ⊚ ⊚ ◯ ◯ ◯ ⊚ ⊚ Δ Δ ◯ Sep (−) ◯ X Δ Δ ◯ X X X ◯◯ X X Δ ⊚: Best in cellular conditions ◯: Good in cellular conditions Δ:Cells with damage X: Cell death Sep: Selenoproteln P fragment

1. An isolated or purified peptide fragment of selenoprotein P,consisting of SEQ ID NO:3, wherein said peptide fragment has celldeath-inhibitory activity.
 2. The isolated or purified peptide fragmentof claim 1, consisting of the amino acid sequence of the formula (I):Lys Arg Cys Ile Asn Gln Leu Leu Cys Lys Leu Pro Thr Asp Ser Glu Leu AlaPro Arg Ser Xaa Cys Cys His Cys Arg His Leu (SEQ ID NO:1) or the aminoacid sequence of the formula (II):Thr Gly Ser Ala Ile Thr Xaa Gln Cys Lys Glu Asn Leu Pro Ser Leu Cys SerXaa Gln Gly Leu Arg Ala Glu Glu Asn Ile (SEQ ID NO:2) wherein Xaa isselenocysteine.
 3. The isolated or purified peptide fragment of claim 1or 2 wherein said peptide fragment is derived from plasma proteins. 4.An additive for cell culture comprising as an active ingredient theisolated or purified peptide fragment of claim
 1. 5. A composition,comprising as an active ingredient the isolated or purified peptidefragment of claim 1; and a suitable adjuvant or solution.